Oxepane isomers of rapamycin useful as immunosuppressive agents

ABSTRACT

A derivative of rapamycin of general formula (I) ##STR1## wherein R 1  and R 2  are selected from the group consisting of hydrogen, acyl, sulfonyl or alkyl or a pharmaceutically acceptable salt thereof, which is by virtue of its immunosuppressive activity is useful in treating transplantation rejection host vs. graft disease, autoimmune diseases, and diseases of inflammation.

This is a division of application Ser. No. 07/822,171, filed Jan. 16,1992, now U.S. Pat. No. 5,221,740.

BACKGROUND OF THE INVENTION

This invention relates to oxepane isomers of rapamycin and a method forusing them in the treatment of transplantation rejection, host vs. graftdisease, autoimmune diseases, diseases of inflammation, solid tumors,and fungal infections.

Rapamycin is a macrocyclic triene antibiotic produced by Streptomyceshygroscopicus, which was found to have antifungal activity, particularlyagainst Candida albicans, both in vitro and in vivo [C. Vezina et al.,J. Antibiot. 28, 721 (1975); S. N. Sehgal et al., J. Antibiot. 28, 727(1975); H. A. Baker et al., J. Antibiot. 31, 539 (1978); U.S. Pat. No.3,922,992; and U.S. Pat. No. 3,993,749].

Rapamycin alone (U.S. Pat. No. 4,885,171) or in combination withpicibanil (U.S. Pat. No. 4,401,653) has been shown to have antitumoractivity. R. Martel et al. [Can. J. Physiol. Pharmacol. 55, 48 (1976)disclosed that rapamycin is effective in the experimental allergicencephalomyelitis model, a model for multiple sclerosis; in the adjuvantarthritis model, a model for rheumatoid arthritis; and effectivelyinhibited the formation of IGE-like antibodies.

The immunosuppressive effects of rapamycin have been disclosed in FASEB3, 3411 (1989). Rapamycin has been shown to be effective in inhibitingtransplant rejection (U.S. patent application Ser. No. 362,544 filedJun. 6, 1989). Cyclosporin A and FK-506, other macrocyclic molecules,also have been shown to be effective as immunosuppressive agents,therefore useful in preventing transplant rejection [FASEB 3, 3411(1989); FASEB 3, 5256 (1989); and R. Y. Calne et al., Lancet 1183(1978)].

Mono- and diacylated derivatives of rapamycin (esterified at the 28 and43 positions) have been shown to be useful as antifungal agents (U.S.Pat. No. 4,316,885) and used to make water soluble prodrugs of rapamycin(U.S. Pat. No. 4,650,803). Recently, the numbering convention forrapamycin has been changed; therefore according to Chemical Abstractsnomenclature, the esters described above would be at the 31- and42-positions.

DESCRIPTION OF THE INVENTION

This invention relates to compounds of general formula I ##STR2##wherein R¹ and R² are selected from the group consisting of hydrogen,acyl, sulfonyl or alkyl or a pharmaceutically acceptable salt thereof.

The compounds of the present invention are oxepane isomers of rapamycinand its derivatives represented in the pyran form by the formula II##STR3## and are prepared from rapamycin or its derivatives by treatmentwith a strong base in an aprotic media or by isolation from the culturemedia in which rapamycin is produced. These compounds display modifiedpharmacodynamic behavior and possess immunosuppressive and/or antifungaland/or antitumor and/or antiinflammatory activity in vivo and/or inhibitthymocyte proliferation in vitro and are therefore useful in thetreatment of transplantation rejection, autoimmune diseases (i.e. lupus,rheumatoid arthritis, diabetes mellitus, multiple sclerosis), Candidaalbicans infections, and diseases of inflammation.

PRIOR ART

The prior art relates to rapamycin itself (U.S. Pat. No. 3,929,992) andto derivatives of rapamycin, wherein the hydroxyl groups at carbonsnumbered 31 and 42 are derivatized to form esters and ethers, along withother modifications of rapamycin. Because rapamycin is unstable toaqueous base solution and the primary mode of decomposition is believedto occur by nucleophilic attack on the electrophilic carbonyl α to theamide of the pyran form of rapamycin; the oxepane analogues, where thecarbonyls are less electrophilic, are more stable and thus ,more potentthan the pyran form of rapamycin and display modified pharmacodynamicbehavior.

An example of the preparation of compounds of formula I, wherein R¹ andR² are hydrogen, is described below.

EXAMPLE 1

Rapamycin (1.0 g, 1.09 mmol) was dissolved in THF (50 mL), cooled to 78°C. and treated with benzyl magnesium chloride (2.5 mL of 2M solution inTHF, 5 mmol). The reaction mixture was warmed to room temperature,stirred for 30 minutes and partitioned between 2N HCl (50 mL) and ethylacetate (60 mL). The organic layer was washed with brine (50 mL), dried(MgSO₄) and concentrated to an oil. The product was chromatographed on aDynamax® 60 C₁₈ column (41.4 mm ID X 30 cm length) using a lineargradient from 100% A (0.1% TFA and 5% acetonitrile in water) to 100% B(pure acetonitrile) over 120 minutes at 25 mL/min. The product elutes in108 minutes.

¹³ C-NMR (DMSO-d₆) d 210.94, 210.71, 209.39, 208.95, 208.30, 208.09,170.17, 169.60, 167.63, 167.45, 139.05, 138.36, 138.09, 137.13, 137.07,136.45, 133.01, 132.29, 130.51, 129.52, 128.52, 127.17 (2C), 126.73,125.35, 124.19, 98.46, 97.95, 85.81, 85.22, 83.83, 83.75, 82.98, 82.39,76.12, 75.92, 73.80, 73.22, 73.15, 73.09, 72.70, 71.69, 57.18, 56.89,56.71, 56.69, 55.24, 54.99, 51.19, 45.12, 42.75 (2C), 42.06, 40.95,40.82, 40.73, 40.33, 39.62, 38.64, 38.52, 37.78, 35.69, 35.49, 35.35,34.46, 33.92, 33.18, 32.94, 32.79 (2C), 32.72, 32.59, 32.18, 31.30,31.13, 30.99, 27.18, 25.99, 24.75, 24.20, 21.52 (2C), 21.28, 20.54,20.46 (2C), 16.94, 16.82 (2C), 16.79 (2C), 15.98, 15.53, 15.43, 14.90,14.74, 13.69, 13.64, 13.43, 13.30, 10.58, 10.30.

The pharmaceutically acceptable salts may be formed from inorganiccations such as sodium, potassium, and the like.

The compounds of this invention possess immunosuppressive and/orantifungal and/or antitumor and/or antiinflammatory activity in vivoand/or inhibit thymocyte proliferation in vitro and are therefore usefulin the prevention and treatment of transplant rejection such as heart,kidney, liver, bone marrow, and skin transplants; graft versus hostdisease; autoimmune and proliferative diseases such as systemic lupuserythematosus, rheumatoid arthritis, type 1 diabetes, multiplesclerosis, glomerular nephritis, Hashimoto's thyroiditis, myasteniagravis, uveitis and psoriasis; fungal infections and diseases ofinflammation such as dermatitis, eczema, seborrhea and inflammatorybowel disease.

The immunosuppressive effects of the compounds of this invention wereevaluated in an in vitro comitogen-induced thymocyte proliferation testprocedure to measure lymphocyte proliferation (LAF) and in two in vivostandard pharmacological test procedures. The first in vivo procedurewas a popliteal lymph node (PLN) test procedure which measured theeffect of compounds of this invention on a mixed lymphocyte reaction andthe second in vivo procedure evaluated the survival time of a pinch skingraft.

The comitogen-induced thymocyte proliferation procedure (LAF) was usedas an in vitro measure of the immunosuppressive effects ofrepresentative compounds. Briefly, cells from the thymus of normalBALB/c mice were cultured for 72 hours with PHA and IL-1 and pulsed withtritiated thymidine during the last six hours. Cells are cultured withand without various concentrations of rapamycin, cyclosporin A, or testcompound. Cells are harvested and incorporated; radioactivity isdetermined. Inhibition of lymphoproliferation is assessed in percentchange in counts per minute from non-drug treated controls. The resultsare expressed by the following ratio:

    ______________________________________                                         ##STR4##                                                                     LAF ASSAY RESULT FOR COMPOUND OF EXAMPLE 1                                    PERCENT CHANGE FROM CONTROL                                                   (mitogen + 0 μM drug added)                                                ______________________________________                                                  1      0.1    10   3    1    0.1  NonLin                            drug conc.                                                                              μM  μM  nM   nM   nM   nM   1C 50                             Drug + IL-1B                                                                            -96    -93    -40  -8   2    4    13.0 nM                           ______________________________________                                    

A mixed lymphocyte reaction (MLR) occurs when lymphoid cells fromgenetically distinct animals are combined in tissue culture. Eachstimulates the other to undergo blast transformation which results inincreased DNA synthesis that can be quantified by the incorporation oftritiated thymidine. Since stimulating a MLR is a function of disparityat Major Histocompatibility antigens, an in vivo popliteal lymph node(PLN) test procedure closely correlates to host vs. graft disease.Briefly, irradiated spleen cells from BALB/c donors are injected intothe right hind foot pad of recipient C3H mice. The drug is given daily,p.o. from Day 0 to Day 4. On Day 3 and Day 4, tritiated thymidine isgiven i.p., b.i.d. On Day 5, the hind popliteal lymph nodes are removedand dissolved, and radioactivity counted. The corresponding left PLNserves as the control for the PLN from the injected hind foot. Percentsuppression is calculated using the non-drug treated animals asallogenic control. ##EQU1##

The second in vivo test procedure is designed to determine the survivaltime of pinch skin graft from male DBA/2 donors transplanted to maleBALB/c recipients. The method is adapted from Billingham R. E. andMedawar P. B., J. Exp. Biol. 28:385-402, (1951). Briefly, a pinch skingraft from the donor is grafted on the dorsum of the recipient as ahomograft, and an autograft is used as control in the same region. Therecipients are treated with either varying concentrations of cyclosporinA as test control or the test compound, intraperitoneally. Untreatedrecipients serve as rejection control. The graft is monitored daily andobservations are recorded until the graft becomes dry and forms ablackened scab. This is considered as the rejection day. The mean graftsurvival time (number of days±S.D.) of the drug treatment group iscompared with the control group.

Based on the results of these standard pharmacological test procedures,the compounds of this invention are useful in the prevention andtreatment of transplant rejection such as heart, kidney, liver, bonemarrow, and skin transplants; graft versus host disease; autoimmune andproliferative diseases such as, systemic lupus erythematosus, rheumatoidarthritis, type 1 diabetes, multiple sclerosis, glomerular nephritis,Hashimoto's thyroiditis, myastenia gravis, uveitis and psoriasis;diseases of inflammation such as dermatitis, eczema, seborrhea andinflammatory bowel disease; and fungal infections.

The compounds of this invention may be administered neat or with apharmaceutical carrier to a mammal in need thereof. The pharmaceuticalcarrier may be solid or liquid.

A solid carrier can include one or more substances which may also act asflavoring agents, lubricants, solubilizers, suspending agents, fillers,gildants, compression aids, binders or tablet-disintegrating agents; itcan also be an encapsulating material. In powders, the carrier is afinely divided solid which is in admixture with the finely dividedactive ingredient. In tablets, the active ingredient is mixed with acarrier having the necessary compression properties in suitableproportions and compacted in the shape and size desired. The powders andtablets preferably contain up to 99% of the active ingredient. Suitablesolid carriers include, for example, calcium phosphate, magnesiumstearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose,methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine,low melting waxes and ion exchange resins.

Liquid carriers are used in preparing solutions, suspensions, emulsions,syrups, elixirs and pressurized compositions. The active ingredient canbe dissolved or suspended in a pharmaceutically acceptable liquidcarrier such as water, an organic solvent, a mixture of both orpharmaceutically acceptable oils or fats. The liquid carrier can containother suitable pharmaceutical additives such as solubilizers,emulsifiers, buffers, preservatives, sweeteners, flavoring agents,suspending agents, thickening agents, colors, viscosity regulators,stabilizers or osmo-regulators. Suitable examples of liquid carriers fororal and parenteral administration include water (partially containingadditives as above, e.g. cellulose derivatives, preferably sodiumcarboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols, e.g. glycols) and their derivatives,and oils (e.g. fractionated coconut oil and arachis oil). For parenteraladministration, the carrier can also be an oily ester such as ethyloleate and isopropyl myristate. Sterile liquid carriers are useful insterile liquid form compositions for parenteral administration. Theliquid carrier for pressurized compositions can be halogenatedhydrocarbon or other pharmaceutically acceptable propellent.

Liquid pharmaceutical compositions which are sterile solutions orsuspensions can be utilized by, for example, intramuscular,intraperitoneal or subcutaneous injection. Sterile solutions can also beadministered intravenously. The compound can also be administered orallyeither in liquid or solid composition form.

Preferably, the pharmaceutical composition is in unit dosage form, e.g.as tablets or capsules. In such form, the composition is sub-divided inunit dose containing appropriate quantities of the active ingredient;the unit dosage forms can be packaged compositions, for example,packeted powders, vials, ampoules, prefilled syringes or sachetscontaining liquids. The unit dosage form can be, for example, a capsuleor tablet itself, or it can be the appropriate number of any suchcompositions in package form. The dosage to be used in the treatmentmust be subjectively determined by the attending physician.

What is claimed is:
 1. A method of treating transplantation rejection,and host vs. graft disease, in a mammal by administering an effectiveamount of a compound of formula (I) ##STR5## wherein R¹ and R² areselected from the group consisting of hydrogen, acyl, sulfonyl or alkylor a pharmaceutically acceptable salt thereof.
 2. A pharmaceuticalcomposition comprising an effective amount of a compound of claim 1 or apharmaceutically acceptable salt thereof and a solid or liquidpharmaceutically acceptable carrier.